Growth/differential factor of the TGF-B family

ABSTRACT

The invention concerns a protein of the TGF-β family, the DNA coding therefor and a pharmaceutical composition containing the protein.

This application is a divisional application of U.S. Patent applicationSer. No. 08/288,508, filed Aug. 10, 1994, now issued as U.S. Pat. No.5,994,094.

The present invention concerns a new growth/differentiation factor ofthe TGF-β family and DNA sequences coding therefor.

The TGF-β family of growth factors which includes BMP-, TGF- andinhibin-related proteins (Roberts and Sporn, Handbook of ExperimentalPharmacology 95 (1990), 419-472) is particularly relevant for a widerange of medical treatment methods and applications. These factors aresuitable in methods which concern wound-healing and tissue regeneration.Furthermore several members from the TGF-β family induce tissue growth,in particular growth of bones, and therefore play a crucial role ininducing the development of cartilage and bones.

Wozney (Progress in Growth Factor Research 1 (1989), 267-280) and Valeet al (Handbook of Experimental Pharmacology 95 (1990), 211-248)describe various growth factors such as those which are related to theBMP group (bone morphogenetic proteins) and the inhibin group. Themembers of these groups show significant structural similarities. Theprecursor of the protein consists of an amino-terminal signal sequence,a propeptide and a carboxy-terminal sequence of about 110 amino acidsthat are cleaved from the precursor and constitute the mature protein.In addition their members are defined by an amino acid sequencehomology. The mature protein contains the most conserved sequences, inparticular seven cysteine residues which are conserved among the familymembers. The TGF-β-like proteins are multifunctional, hormonally activegrowth factors. They also have related biological activities such aschemotactic attraction of cells, promotion of cell differentiation andtissue-inducing capabilities such as cartilage-inducing andbone-inducing capabilities. The U.S. Pat. No. 5,013,649 discloses DNAsequences that code for osteo-inductive proteins that are denoted BMP-2and the U.S. patent applications Ser. No. 179 101 and 170 197 disclosethe BMP proteins BMP-1 and BMP-3. Moreover many types of cells are ableto synthesize TGF-β-like proteins and practically all cells have TGF-βreceptors.

On the whole these proteins show differences in their structure whichleads to significant variations in their exact biological function. Inaddition they are found in a wide range of different types of tissue andat various stages of development. As a result they can exhibitdifferences with regard to their exact function e.g. the requiredcellular physiological environment, their life-span, their target sites,their requirements for auxiliary factors and their stability againstdegradation. Thus, although a multitude of proteins have been describedthat exhibit a tissue-inductive and in particular osteo-inductivepotential, their natural functions in the organism and—moresignificantly—their medical relevance still have to be investigated indetail. It is thought to be highly probable that members of the TGF-βfamily are present that are still unknown which are important forosteogenesis or the differentiation/induction of other types of tissue.A major difficulty in the isolation of these new TGF-β-like proteins is,however, that their functions cannot yet be described exactly enough todevelop highly discriminating bioassays. On the other hand the expectednucleotide sequence homology to other members of the family is too lowto enable a screening by classical nucleic acid hybridizationtechniques. Nevertheless the further isolation and characterization ofnew TGF-β-like proteins is urgently required in order to provide furtherinducing and differentiation-promoting proteins that fulfil all thedesired medical requirements. These factors could be used medically inthe healing of lesions and the treatment of degenerative diseases ofbones and/or other types of tissue such as the kidney or the liver.

A nucleotide and amino acid sequence for the TGF-β protein MP-52 isstated in the Patent Application PCT/EP93/00350 in which the sequencecorresponding to the mature peptide and a major portion of the sequencecorresponding to the propeptide of MP-52 are given. The completesequence of the propeptide MP-52 is not disclosed.

The object on which the present invention is based is to provide DNAsequences that code for new members of the TGF-β protein family withmitogenic and/or differentiation-inductive e.g. osteo-inductivepotential. The object of the present invention was therefore inparticular to provide the complete DNA and amino acid sequence of theTGF protein MP-52.

This object is achieved by a DNA molecule that codes for a protein ofthe TGF-β family and which comprises

(a) the part coding for the mature protein and if desired furtherfunctional parts of the nucleotide sequence shown in SEQ ID NO. 1,

(b) a nucleotide sequence corresponding to a sequence from (a) withinthe scope of the degeneracy of the genetic code,

(c) an allelic derivative of a nucleotide sequence corresponding to oneof the sequences from (a) and (b) or

(d) a sequence hybridizing with one of the sequences from (a), (b) or(c) provided that a DNA molecule according to (d) contains at least thepart coding for a mature protein from the TGF-β family.

Further embodiments of the present invention concern the subject matterof the further claims. Other features and advantages of the inventioncan be derived from the description of the preferred embodiments andfigures. The sequence protocols and figures are now briefly described.

SEQ ID NO. 1 shows the complete nucleotide sequence of the DNA codingfor the TGF-β protein MP-52. The ATG start codon starts with nucleotide640. The start of the mature protein begins after nucleotide 1782.

SEQ ID NO. 2 shows the complete amino acid sequence of the TGF-β proteinMP-52 which was derived from the nucleotide sequence shown in SEQ ID NO.1.

FIG. 1 shows a comparison between the amino acid sequence of MP-52 andseveral members of the BMP protein family starting with the first of theseven conserved cysteine residues. * denotes that the amino acid is thesame in all compared proteins; + denotes that the amino acid correspondsin at least one of the proteins compared to MP-52.

FIG. 2 shows the nucleotide sequences of the oligonucleotide primersthat were used in the present invention and a comparison of thesesequences with known members of the TGF-β family. M denotes A or C, Sdenotes C or G, R denotes A or G and K denotes G or T. 2 a shows thesequence of the primer OD, 2 b shows the sequence of the primer OID.

FIG. 3 discloses a Western blot indicating the production of MP52 usingvaccinia viruses as expression systems.

FIG. 4 discloses a schematic view of the plasmid pABWN.

FIG. 5 discloses a section of an implant (matrix-bound MP52, 26 daysafter implantation) stained according to von Kossa. Mineralized tissuein black is clearly distinguished from the surrounding muscle tissue.

FIG. 6 discloses a partial cross-section view of the implant of FIG. 5,but stained according to Masson-Goldner.

The present invention encompasses at least the part coding for themature protein and if desired further functional parts of the nucleotidesequence shown in SEQ ID NO. 1 as well as sequences that correspond tothis sequence within the scope of the degeneracy of the genetic code andallelic derivatives of such sequences. In addition the present inventionalso encompasses sequences that hybridize with such sequences providedthat such a DNA molecule completely contains at least the part codingfor the mature protein of the TGF-β family.

The term “functional part” within the sense of the present inventiondenotes a protein part which is capable of acting for example as asignal peptide, propeptide or as a mature protein part i.e. it fulfilsat least one of the biological functions of the natural protein parts ofMP-52.

The region coding for the mature part of the protein extends fromnucleotides 1783-2142 of the sequence shown in SEQ ID NO. 1. If desired,the DNA molecule can also comprise further functional parts of thesequence shown in SEQ ID NO. 1, namely the nucleotide sequences codingfor the signal or/and propeptide part. It is particularly preferred thatthe DNA molecule comprises the sequence for the signal and thepropeptide part and the part of the mature protein i.e. nucleotides640-2142 of the sequence shown in SEQ ID NO. 1. On the other hand theDNA molecule can also comprise functional signal or/and propeptide partsfrom other proteins in addition to the part coding for the matureprotein, in particular from other proteins of the TGF-β family e.g. theabove-mentioned BMP proteins. The respective nucleotide sequences aregiven in the references mentioned above to the disclosure of whichreference is hereby being made.

Moreover the present invention also encompasses a DNA molecule asdefined above that contains a non-coding intron sequence betweennucleotides 1270 and 1271 of the sequence shown in SEQ ID NO. 1. Thisintron sequence is contained in the plasmid SKL 52 (H3) MP12 which isdeposited at the DSM and has the genomic nucleic acid sequence of MP-52.

The invention also encompasses the cDNA sequence of the MP-52 proteincoded by the phage λ15.1. This sequence starts with nucleotide 321 ofSEQ ID NO. 1.

Although the allelic, degenerate and hybridizing sequences which areencompassed by the present invention have structural differences due toslight changes in their nucleotide or/and amino acid sequence, theproteins coded by such sequences still essentially have the same usefulproperties that enable their use in basically the same medicalapplications.

The term “hybridization” according to the present invention means theusual hybridization conditions, preferably conditions with a saltconcentration of 6×SSC at 62 to 66° C. followed by a one hour wash with0.6×SSC, 0.1% SDS at 62 to 66° C. It is particularly preferred that theterm “hybridization” denotes stringent hybridization conditions with asalt concentration of 4×SSC at 62 to 66° C. followed by a one hour washwith 0.1×SSC, 0.1% SDS at 62 to 66° C.

Preferred embodiments of the present invention are DNA sequences asdefined above that are obtainable from vertebrates, preferably mammalssuch as pigs, cows and rodents such as rats or mice and in particularfrom primates such as humans.

A particularly preferred embodiment of the present invention is thesequence denoted MP-52 shown in SEQ ID NO. 1. The transcripts of MP-52were obtained from embryonic tissue and code for a protein which has aconsiderable amino acid homology to the mature portion BMP-like proteins(see FIG. 1). The protein sequences of BMP2 (=BMP2A) and BMP4 (=BMP2B)are described by Wozney et al., Science 242 (1988), 1528-1534. Thecorresponding sequences of BMP5, BMP6 and BMP7 are described by Celesteet al., Proc. Natl. Acad. Sci. USA 87 (1990), 9843-9847. Several typicalsequence homologies which are specific for known BMP sequences have alsobeen found in the propeptide part of MP-52 whereas other parts of theprecursor part of MP-52 exhibit considerable differences to BMPprecursors.

In addition the present invention concerns a vector that contains atleast one copy of a DNA molecule according to the invention. The DNAsequence according to the invention is preferably operatively linked toan expression control sequence in such a vector. Such vectors aresuitable for the production of TGF-β-like proteins in stably ortransiently transformed cells. Various animal, plant, fungal andbacterial systems can be used for the transformation and subsequentculture. The vectors according to the invention preferably containsequences necessary for replication in the host cell and areautonomously replicable. Furthermore the use of vectors is preferredthat contain selectable marker genes which can be used to detecttransformation of a host cell.

In addition the invention concerns a host cell which is transformed witha DNA according to the invention or with a vector according to theinvention. Examples of suitable host cells include various eukaryoticand prokaryotic cells such as E. coli, insect cells, plant cells,mammalian cells and fungi such as yeast.

In addition the invention concerns a protein of the TGF-β family whichis coded by a DNA sequence according to claim 1. The protein accordingto the invention preferably has the amino acid sequence shown in SEQ IDNO. 2 or if desired functional parts thereof and exhibits biologicalproperties such as tissue-inductive in particular osteo-inductive or/andmitogenic capabilities that may be relevant for a therapeuticapplication. The above-mentioned characteristics of the protein can varydepending on the formation of homodimers or heterodimers. Suchstructures may also prove to be suitable for clinical applications.

The biological properties of the proteins according to the invention, inparticular the mitogenic and osteo-inductive potential can be determinedfor example in assays according to Seyedin et al., PNAS 82 (1985),2267-2271 or Sampath and Reddi, PNAS 78 (1981), 7599-7603.

Furthermore the present invention concerns a process for the productionof a protein of the TGF-β family which is characterized in that a hostcell transformed with a DNA according to the invention or with a vectoraccording to the invention is cultured and the TGF-β protein is isolatedfrom the cell or/and culture supernatant. Such a process comprisesculturing the transformed host cell in a suitable culture medium andpurifying the TGF-β-like protein produced. In this manner the processenables the production of an adequate amount of the desired protein foruse in medical treatment or in applications using cell culturetechniques which require growth factors. The host cell can be abacterium such as Bacillus or E. coli, a fungus such as yeast, a plantcell such as tobacco, potato or arabidopsis or an animal cell and inparticular a vertebrate call line such as MoCOS or CHO cell lines or aninsect cell line.

Yet another subject matter of the present invention is the provision ofpharmaceutical compositions which contain a pharmaceutically activeamount of a TGF-β-like protein according to the invention as the activesubstance. If desired, such a composition comprises a pharmaceuticallyacceptable carrier substance, auxiliary substance, diluent or filler.Such a pharmaceutical composition can be used in wound-healing andtissue regeneration as well as in the healing of damage to bones,cartilage, connective tissue, skin, mucous membranes, epithelium orteeth and in dental implants either alone or in combination with otheractive substances e.g. other proteins of the TGF-β family or growthfactors such as EGF (epidermal growth factor) or PDGF (platelet derivedgrowth factor). Moreover such a pharmaceutical composition can be usedin the prevention of diseases such as the prevention of osteoporosis andarthrosis.

Another possible clinical application of the TGF-β protein according tothe invention is to use it as a suppressor of an immunoreaction toprevent rejection of organ transplants or its application in connectionwith angiogenesis. The pharmaceutical composition according to theinvention can also be used prophylactically or in cosmetic surgery. Inaddition administration of the composition is not limited to humans butcan also encompass animals and in particular pets.

Finally the present invention also concerns an antibody that bindsspecifically to the proteins according to the invention or such anantibody fragment (e.g. Fab or Fab′). Processes for the production ofsuch a specific antibody or antibody fragment are part of the generaltechnical knowledge of an average person skilled in the art. Such anantibody is preferably a monoclonal antibody. Such antibodies orantibody fragments may also be suitable for diagnostic methods.

It is intended to elucidate the invention further by the followingexample.

EXAMPLE 1 Isolation of MP-52

1.1 Total RNA was isolated from human embryonic tissue (8 to 9 weeksold) according to the method of Chirgwin et al., Biochemistry 18 (1979),5294-5299. Poly(A+) RNA was separated from the total RNA by oligo (dT)chromatography according to the manufacturer's instructions (StratagenePoly (A) Quick columns).

1.2 For the reverse transcription reaction 1 to 2.5 μg poly (A+) RNA washeated for 5 minutes to 65° C. and quickly cooled on ice. The reactionmixture contained 27 U RNA-Guard (Pharmacia), 2.5 μg oligo (dT)12-18(Pharmacia), 5×buffer (250 mmol/l Tris/HCl, pH 8.5, 50 mmol/l MgCl₂, 50mmol/l DTT, 5 mmol/l of each dNTP, 600 mmol/l KCl) and 20 U AMV reversetranscriptase (Boehringer Mannheim) per μg poly (A+) RNA. The reactionmixture (25 μl) was incubated for 2 hours at 42° C.

1.3 The deoxynucleotide primers OD and OID shown in FIG. 2 were preparedon an automatic DNA synthesizer (Biosearch). The purification wascarried out by denaturing polyacrylamide gel electrophoresis andisolating the main bands from the gel by means of isotachophoresis. Theoligonucleotides were designed by comparing the nucleic acid sequencesof known members of the TGF-β family and selecting regions with thehighest conservation. A comparison of this region is shown in FIG. 2. Inorder to facilitate cloning both nucleotides contained EcoRI restrictionsites and OD additionally contained a NcoI restriction site at its 5′terminus.

1.4 cDNA corresponding to 20 ng poly (A+) RNA was used as the startingmaterial in the PCR reaction. The reaction was carried out in a volumeof 50 μl and contained 1×PCR buffer (16.6 mmol/l (NH₄)₂SO₄, 67 mmol/lTris/HCl pH 8.8, 2 mmol/l MgCl₂, 6.7 μmol/l EDTA, 10 mmol/lβ-mercapto-ethanol, 170 μg/ml bovine serum albumin (Gibco), 200 μmol/lof each dNTP (Pharmacia), 30 pmol of each oligonucleotide (OD and OID)and 1.5 U Taq polymerase (AmpliTaq, Perkin Elmer Cetus). The reactionmixture was overlayed with paraffin and 40 PCR cycles were carried out.The products of the PCR reaction were purified by phenol/chloroformextraction and concentrated by ethanol precipitation.

1.5 The PCR reaction product was cleaved with the restriction enzymesSphI (Pharmacia) and AlwNI (Biolabs) according to the manufacturer'sinstructions.

1.6 The products of the restriction cleavage were fractionated byAgarose gel electrophoresis. After staining with ethidium bromide,uncleaved amplification products were cut out of the gel and isolated byphenol extraction. The DNA obtained was subsequently purified twice byphenol/chloroform extraction.

1.7 After an ethanol precipitation, a quarter or a fifth of the isolatedDNA was reamplified using the same conditions as for the primaryamplification except that the number of cycles was reduced to 13. Thereamplification products were purified, cleaved with the same enzymes asabove and the uncleaved products were isolated from Agarose gels aselucidated above for the amplification products. The reamplificationstep was repeated twice.

1.8 After the last isolation from the gel, the amplification productswere cleaved by 4 units EcoRI (Pharmacia) under the conditionsrecommended by the manufacturer. A fourth of the restriction mixture wasligated into the vector pBluescriptII SK+ (Stratagene) cleaved withEcoRI. 24 clones were analysed further by means of sequencing afterligation. The sample cleaved with AlwNI and SphI resulted in a newsequence that was denoted MP-52. The other clones mainly contained BMP6sequences and one contained a BMP7 sequence.

The clone was completed up to the 3′ end of the cDNA according to themethod described in detail by Frohmann (Amplifications, published byPerkin-Elmer Corp., Issue 5 (1990), pp 11-15). The same embryonic mRNAthat had been used to isolate the first fragment of MP-52 was reversallytranscribed as described above. The amplification was carried out usingthe adapter primer (AGAATTCGCATGCCATGGTCGACG) (SEQ ID NO:3) of the MP-52sequence. The amplification products were reamplified using anoverlapping adapter primer (ATTCGCATGCCATGGTCGACGAAG) (SEQ ID NO:5) andan overlapping internal primer (GGAGCCCACGAATCATGCAGTCA) (SEQ ID NO:6)of the MP-52 sequence. After restriction cleavage with NcoI thereamplification products were cloned and sequenced Into a vector thatwas cleaved in the same way (pUC 19 (Pharmacia No. 27-4951-01) having amodified multiple cloning site which contains a single Ncol restrictionsite) and sequenced. The clones were characterized by their sequenceoverlapping at the 3′ end of the known MP-52 sequence. One of these wasused as a probe to screen a human genomic gene bank (Stratagene No.946203) according to a method described in detail by Ausubel et al.(Current Protocols in Molecular Biology, published by Greene PublishingAssociates and Wiley-lnterscience (1989)). One phage (λ2.7.4) wasIsolated from 8×10⁵ λ phages which contained an insertion of about 20 kband which is deposited at the DSM under the depository number 7387. Thisclone contains further sequence information at the 5′ end in addition tothe sequence isolated from mRNA by the described amplification methods.

For the sequence analysis a HindIII fragment of about 7.5 kb wassubcloned into a vector cleaved in the same manner (Bluescript SK,Stratagene No. 212206). This plasmid denoted SKL 52 (H3) MP12 was alsodeposited at the DSM under the depository number 7353. The sequenceinformation shown in SEQ ID No. 1 was derived from the phage λ2.7.4. TheATG at position 640 is the first ATG within the reading frame (a stopcodon occurs at position 403). Based on the sequence data it may beassumed that this is the start codon for the translation.

The genomic DNA contains an intron of about 2 kb between base paris 1270and 1271 of SEQ ID NO:1. The sequence of the intron is not shown. Thecorrectness of the splicing site was confirmed by sequencing anamplification product which was derived from cDNA containing thisregion. These sequence informations were obtained using a slightlymodified method which is described in detail by Frohmann(Amplifications, published by Perkin-Elmer Corporation, Issue 5 (1990),pp 11-15). The same embryonic RNA that was also used to isolate the 3′end of MP-52 was reverse transcribed using an internal primer orientatedin the 5′ direction of the MP-52 sequence (ACAGCAGGTGGGTGGTGTGGACT) (SEQID NO:7). A polyA tail was attached to the 5′ end of the first cDNAstrand using terminal transferase. A two-step amplification was carriedout, firstly by using a primer composed of oligo dT and an adaptersequence (AGAATTCGCATGCCATGGTCGACGAAGC(T16)) (SEQ ID NO:8) and secondlyan adapter primer (AGAATTCGCATGCCATGGTCGACG) (SEQ ID NO:3) and aninternal primer (CCAGCAGCCCATCCTTCTCC) (SEQ ID NO:9) from the MP-52sequence. The amplification products were reamplified using the sameadapter primer and an overlapping internal primer(TCCAGGGCACTAATGTCAAACACG) (SEQ ID NO:10) from the MP-52 sequence.Subsequently the reamplification products were reamplified using anoverlapping adapter primer (ATTCGCATGCCATGGTCGACGAAG) (SEQ ID ON:5) andan overlapping internal primer (ACTAATGTCAAACACGTACCTCTG) (SEQ ID NO:11)from the MP-52 sequence. The final reamplification products were clonedwith blunt ends into a vector (Bluescript SK, Stratagene No. 212206)which had been cleaved with EcoRV. The clones were characterized bytheir sequence overlapping with the DNA of λ2.7.4.

In addition a cDNA bank—produced from RNA of human fibroblasts andcloned into λgt10—was screened. In this process 2×10⁶ phages were testedusing a ca. 1 kb fragment of genomic MP-52 DNA (2nd exon up to theHindIII restriction site in the 3′ untranslated region) as a radioactiveprobe. 17 mixed plaques were picked out and were checked by PCR usingprimers from the 5′ and 3′ region of the MP-52 sequence. Subsequently 8phage plaques were selected and isolated. cDNA was isolated from thephage by an EcoRI partial cleavage and cloned into the Bluescript vectorthat was also cleaved with EcoRI.

Sequencing of one of the resulting plasmids SK52L15.1MP25 showed thatthe longest phage (15.1) starts at nucleotide No. 321 of SEQ ID No. 1.In addition the splicing position (nucleotide 1270) was confirmed by thesequencing.

The plasmid SKL 52 (H3) MP12 was deposited on 10th Dec. 1992 at the DSM(“Deutsche Samnlung von Mikroorganismen und Zellkulturen, MascheroderWeg 1b, 38124 Braunschweig) under the depository number 7353.

The phage λ2.7.4 was deposited on 13th Jan. 1993 at the DSM under thedepository number 7387.

The plasmid SK52L15.1MP25 was deposited on 16th Jul. 1993 at the DSMunder the depository number 8421.

EXAMPLE 2 Expression of MP52

Various systems were checked for the expression of MP52. The use ofVaccinia viruses as an expression system is described in detail andcapable of being reproduced by a person skilled in the art in CurrentProtocols in Molecular Biology (Ausubel et al., Greene PublishingAssociates and Wiley-Interscience, Wiley & Sons), abbreviated CP in thefollowing, in chapter 16 unit 16.15-16.18. The system is based on thefact that foreign DNA can be integrated by homologous recombination intothe genome of the Vaccinia virus using certain vectors. For this purposethe vector used contains the TK (thymidine kinase) gene from theVaccinia genome. In order enable selection for recombinant viruses thevector in addition contains the E. coli xanthine-guanine phosphoribosyltransferase gene (gpt) (Falkner et al., J. Virol. 62 (1988), 1849-1854).The cDNA with the entire region coding for MP52 was cloned into thisvector. The cDNA comes from plasmid SK52L15.1MP25 (DSM, depositorynumber 8421) which was, however, firstly deleted and subcloned in orderto remove a large portion of the 5′ untranslated region. For this theplasmid SK52L15.1MP25 was linearized with SalI and the 5′ end wasdeleted stepwise with the ExoIII/mung bean kit (Stratagene #200330)according to the manufacturer's instructions. After restriction withBamHI, the MP52 cDNAs that had been deleted to different extents wereseparated from the residual vector and isolated by an agarose gel andsubcloned (pSK52s) according to standard methods (Sambrook et al.,Molecular cloning, second edition, Cold Spring Harbor Laboratory Press1989) in a pBluescriptII SK vector (Stratagene #212206) restricted withEcoRV and BamHI. All restrictions were carried out according to themanufacturer's instructions. Sequencing with Sequenase (USB/Amersham#70770) yielded inter alia a clone which starts with nucleotide 576 inSEQ ID NO. 1 (64 base pairs distant from the start codon). The cDNAinsert was isolated from this by restriction with SalI and SacI andcloned into the likewise cleaved vector for recombination in thevaccinia virus system. The resulting plasmid (pBP1MP52s) was depositedon 24th May 1994 at the DSM (deposit number 9217) and used for theproduction of recombinant Vaccinia viruses. For this up to 80% confluent143B cells (HuTk, ATCC CRL 8303) in 35 mm culture dishes were infectedwith Vaccinia wild-type virus in 2 ml PBS for 30 minutes at roomtemperature while shaking occasionally (1 virus per 10 cells). Afteraspirating the supernatant and adding 2 ml culture medium (MEM, GibcoBRL #041-01095), it was incubated for 2 hours at 37° C. The medium wassubsequently removed and transformation of these cells was achieved with100 ng pBP1MP52s, 2 μg carrier DNA (calf thymus, Boehringer Mannheim#104175) and 10 μl Lipofectin (Gibco BRL #18292-011) in 1 ml MEM for 15hours at 37° C. After addition of 1 ml MEM containing 20% FCS (Gibco BRL#011-06290), they were incubated for a further 24 hours at 37° C. andsubsequently the lysed cells were frozen.

The gpt selection for xanthine-guanine phosphoribosyl transferase andisolation and amplification of individual recombinant viruses wasessentially carried out as described in unit 16.17 of CP except thatRK13 cells (ATCC CCL 37) were used.

Integration of MP52 cDNA into the virus genome was confirmed by dot blotand Southern blot analysis (CP unit 16.18). A recombinant virus was usedfor expression analyses in the cell line 143B (HuTk-, ATCC CRL 8303,human). Confluent cells were infected for 45 minutes at 37° C. with anumber of viruses corresponding to the number of cells and subsequentlyadded to the respective culture medium (MEM, Gibco BRL #041-01095)containing 10% FCS and penicillin/streptomycin (1:500, Gibco BRL#043-05140H). After 6 hours at 37° C., the medium was removed, the cellswere washed twice with e.g. HBSS (Gibco BRL #042-04180M) and productionmedium (e.g. MEM) without FCS was added. After 20 to 22 hours ofproduction the cell supernatant was collected. The expression wasanalysed by means of Western blots according to standard methods (CPunit 10.8). For this the proteins from 100 to 500 μl cell culturesupernatant were precipitated by addition of an equivalent volume ofacetone and incubating for at least one hour on ice and centrifuged.After resuspending the pellet in application buffer (7 M urea, 1% SDS, 7mM sodium dihydrogen phosphate, 0.01% bromophenol blue and if desired 1%β-mercaptoethanol) separation was carried out in 15% polyacrylamidegels. A prestained protein molecular weight standard (Gibco BRL#26041-020) was used as the marker proteins. Transfer onto a PVDFmembrane (Immobilon #IPVH00010) and blocking the membrane were carriedout according to standard methods.

In order to detect MP52 on the membrane, polyclonal antibodies againstMP52 had been produced in chickens as well as in rabbits. For this themature part of MP52 with 6 histidines at the N-terminus was expressed inE. coli and purified as described for example in Hochuli et al.(BIO/Technology, Vol. 6, 1321-1325 (1988)). Both antibodies enable thespecific detection of expression of MP52 wherein dimeric MP52 is lessefficiently recognized than monomeric. Chicken antibodies were used forthe Western blot in FIG. 3 that had been specifically purified by meansof PEG precipitation (Thalley et al., BIO/Technology vol. 8 934-938(1990)) and by means of membrane-bound antigen (mature MP52 containing 6histidines) (18.17 in Sambrook et al., Molecular cloning, secondedition, Cold Spring Harbor Laboratory Press 1989). Anti-chicken IgGwith coupled alkaline phosphatase (Sigma A9171) was used as the secondantibody. The detection was carried out using the Tropix Western-Lightprotein detection kit (Serva #WL10RC) according to the manufacturer'sinstructions.

The Western blot in FIG. 3 shows that MP52-specific bands only occurwith the recombinant viruses but not with the wild-type viruses (withoutintegrated foreign-DNA). The expression of MP52 results in a secretedprotein having an apparent molecular weight of about 25 kDa in the gelunder non-reducing conditions. The protein migrates in the gel at 14 to15 kDa under reducing conditions. These results show that MP52 isexpressed as a dimeric mature protein. The weak bands appearing in theregion above 60 kDa that occur in the Western blot are probably residuesof the uncleaved precursor proteins. The migration properties alsoconfirms the theoretical molecular weights that can be derived from SEQID NO. 2 according to which mature, monomeric MP52 has a size of 13.6kDa.

It has been proven to be possible to express MP52 and cleave theprecursor protein to mature MP52 in various cell lines. C127 (ATCC CRL1616, mouse), BHK21 (ATCC CCL 10, hamster), MRC-5 (ATCC CCL 171, human)and 3T6-Swiss albino (ATCC CCL 96, mouse) cells were tested.

Expression and cleavage to form mature MP52 was also demonstrated in afurther eukaryotic expression system. For this cDNA from MP52 (startingwith nucleotide 576) was cloned into the expression plasmid pSG5(Stratagene #216201). The plasmid pSK52s was restricted with ClaI andXbaI and the protruding ends of the MP52 insert were made blunt by T4polymerase treatment. Cloning into the vector pSG5, that had beenrestricted with EcoRI and likewise blunt ended by T4 polymerasetreatment, was carried out according to standard methods. All enzymaticreactions were carried out according to the instructions of themanufacturer. Correct orientation of the MP52 insert was ensured byrestriction analysis and sequencing with the T7 primer (Stratagene#300302). The resulting plasmid pSG52s (deposited on 17.05.94 at the DSMwith the deposit number DSM 9204) can be cotransformed with a vectorthat codes for a selectable marker such as e.g. the gene for G418resistance in order to obtain stable cell lines. For this purpose pSG52swas cotransformed with the plasmid p3616 (deposited on 17.05.94 at theDSM with the deposit number DSM 9203) in L929 cells (ATCC CCL1, mouse)using Lipofectin (Gibco BRL #18292-011) according to the manufacturer'sinstructions. Selection with G418 was carried out according to methodsfamiliar to a person skilled in the art (CP, unit 9.5) and it resultedin a cell line that produced detectable mature MP52 in the Western blot.

A further expression vector for MP52 was produced using the plasmidpABWN (Niwa et al., Gene 108 (1991), 193-200 and FIG. 4) which wasprovided by Dr. Miyazaki.

For this the HindIII fragment from plasmid pSK52s that starts withnucleotide 576 in SEQ ID NO. 1, was isolated and the protruding endswere made blunt by treatment with Klenow fragment. A Not I restrictioncleavage site was introduced at both ends of the fragment by ligation ofthe adapter.

Adapter: AGCGGCCGCT (SEQ ID NO:12)

TCGCCGGCGA (SEQ ID NO: 41)

Vector pABWN was restricted with XhoI, also treated with the Klenowfragment and dephosphorylated with intestinal alkaline phosphatase fromthe calf (Boehringer Mannheim). The same phosphorylated adapter wasligated on so that an insertion of the MP52 fragment after restrictionwith Notl into the generated Not I cleavage site of the vector was nowpossible. The expression vector that results is subsequently denotedHindIII-MP52/pABWN. All the reactions carried out for the cloning werecarried out according to standard methods (e.g. CP units 3.16).

HindIII-MP52/pABWN was transfected in L cells* (mouse fibroblasts) andstable transformants were established therefrom.

For this 4 μg in each case of the plasmids (HindIII-MP52/pABWN or pABWN)were transfected in 5×10⁵ L cells in a 6 cm culture dish using 20 μllipofectAMINE reagent (Gibco BRL #18324-012). For this solution A (4 μgof the respective DNA in 200 μl OPTI-MEM I (Gibco BRL #31985)) wascarefully mixed with solution B (20 μl lipofectAMINE reagent in 200 μlOPTI-MEM I) and incubated for 45 minutes at room temperature to form theDNA liposome complex. In the course of this the cells were washed oncewith 2 ml OPTI-MEM I. For each transfection, 1.6 ml OPTI-MEM I was addedto the vessel with the DNA liposome complex. The solution was carefullymixed and the washed cells were overlayed therewith. The cells wereincubated with the dilute complex for 5 hours at 37° C. in an CO₂incubator. After the incubation 2 ml DMEM (Gibco BRL, Dulbecco'smodified eagle medium)/20% FCS was added. 24 hours after thetransfection, the medium was replaced with fresh DMEM/10% FCS. 48 hoursafter the start of transfection, the cells were transferred into a 10 cmculture dish. 72 hours after the start of the transfection, the G418selection was started at a concentration of 800 μg/ml. The stable clonesappeared after 1 to 2 weeks.

5 ml conditioned DMEM with or without FCS was obtained from confluenttransformants which had been grown for 3 days in a 10 cm culture dish.The two different cell culture supernatants. (HindIII-MP52/pABWN andpABWN) of transfected cells as well as cell lysates were examined byWestern blot. In this process mature MP52 was found in conditionedmedium as well as in cell lysates of cells transfected withHindIII-MP52/pABWN. The clones were further cloned and cells producingMP52 were each selected after Western blot analysis. Estimations fromWestern blot analyses yielded a MP52 production of up to 1 mg/l.

EXAMPLE 3 Biological Activity of MP52

Several experiments were carried out in vitro and in vivo in order todetect the biological activity of MP52 and to prove the usefulness ofthis invention for medical applications for the prevention and/ortreatment of bone diseases.

1. In Vitro Assays

1.1

Since an increase in glycosaminoglycan (GAG) synthesis is described inchondrocytes after stimulation with TGF-β (Hiraki et al., Biochimica etBiophysica Acta 969 (1988), 91-99), it was examined whether MP52 alsohas this influence. The chondrogenic activity of MP52 was tested inprimary cultures from foetal rat extremities using the cell culturesupernatants (DMEM containing 10% FCS) of L cell transformants producingMP52 (transfected with HindIII MP52/pABWN).

The four extremities of 16-day-old rat foetuses were used for this.After trypsination, the cells obtained in F-12 medium (Nutrient mixtureHam's F-12, Gibco BRL #21700) containing 10% FCS were plated out at3×10⁵ cells on 24-well plates coated with collagen type I and culturedfor ca. 2 days until confluence. 56 μl conditioned medium (CM) ofHindIII-MP52/pABWN-L cell transfectants, of pABWN-L cell transfectantsor only medium (DMEM containing 10% FCS) was added to 500 μl culturemedium in each case (F-12 medium containing 10% FCS). F-12 mediumcontaining 10% FCS as well as the respective additives was used over aperiod of 0, 3, 6 and 9 days. The medium containing the respectiveadditives was exchanged every three days. Afterwards the culture wascultured for a further 2 days in F-12 medium without FCS in the presenceof the respective additives (conditioned medium or control medium) andthen ³⁵S sulfate was added for 6 hours. ³⁵S incorporated intopolysaccharides was measured after pronase E digestion and precipitationas described in Hiraki et al. (Biochimica et Biophysica Acta 969 (1988),91-99).

TABLE 1 Radioactivity (cpm/well) CM from DMEM (10% CM from HindIII-Number of FCS) from pABWN-L MP52/pABWP-L days of control L cell cellincubation cells transfectants transfectants  2 3720 ± 114 3865 ± 1204879 ± 422   5 4188 ± 135 4154 ± 29  6223 ± 275*   8 3546 ± 160 3310 ±115 9890 ± 1260* 11 3679 ± 218 3633 ± 167 7520 ± 160*  Values relate to± S.E.M. for 3 or 4 culture mixtures *: p < 0.01 vs DMEM CM from pABWN-Lcell transfectants (Scheffe's multiple t-test)

As shown in Table 1, the cell culture supernatants of the transfectantsproducing MP52 significantly stimulate GAG synthesis in comparison topure culture medium (DMEM containing 10% FCS) or to the cell culturesupernatant from L cells transfected with PABWN. This shows that MP52can stimulate differentiation of chondrocytes.

1.2

An effect which has been described for some members of the BMP family isthe stimulation of alkaline phosphatase (ALP) activity in osteoblasts.The clonal rat cell line ROB-C26 (C-26) is among the osteoblasts at arelatively early stage of maturation (Yamaguchi et al, Calcif. TissueInt. 49 (1991), 221-225). The capability of stimulating ALP activity isdescribed for osteoinductive proteins such as e.g. BMP2 by Yamaguchi etal. (J. Cell Biol. 113 (1991), 681-687).

The influence of MP52 on C26 cells was examined as follows: C26 cellswere plated out at 3×10⁴ cells per well in a 24-well plate and culturedin α-MEM (Gibco BRL)/10% FCS until confluence. 56 μl of the cell culturesupernatant from L cell transfectants producing MP52 (HindIII-MP52/pABWN) or of the cell culture supernatant from pABWN-L celltransfectants or only of the cell culture supernatant (DMEM containing10% FCS) from L cells was added to 500 μl of the C-26 cell culturemedium. A change of medium with the respective additives was carried outevery three days. The ALP activity in the cell extracts was determinedafter 0, 3, 6, 9 and 12 days with the aid of standard techniques basedon p-nitrophenyl phosphate as the substrate as described for example byTakuwa et al. (Am. J. Physiol. 257 (1989), E797-E803).

TABLE 2 ALP activity (nmol/min) per well CM from DMEM (10% CM fromHindIII- Number of FCS) from pABWN-L MP52/pABWP-L days of control L cellcell incubation cells transfectants transfectants  0  41.8 ± 2.8  41.8 ±2.8 41.8 ± 2.8   3 136.3 ± 3.7 125.8 ± 2.3 181.3 ± 14.2*  6 129.0 ± 7.8119.3 ± 6.4 258.0 ± 8.3*   9 118.4 ± 3.7 110.1 ± 2.8 258.4 ± 10.6* 12121.2 ± 3.2 125.3 ± 6.0 237.8 ± 11.0* Values relate to ± S.D. for 4culture mixtures. *: p < 0.01 vs DMEM and CM from pABWN-L celltransfectants (Scheffe's multiple t-test)

As shown in Table 2, addition of MP52 leads to a significant increase inALP activity compared to pure DMEM/10% FCS medium and medium frompABWN-infected L cells. This result shows that MP52 cannot only causechondrocytes to differentiate but can also lead to the differentiationand maturation of osteoblasts.

A further osteoblast cell line (MC3T3-E1, mouse) that shows an increasein the ALP activity by treatment with BMP-2 as described by Takuwa etal. (Biochem. Biophys. Res. Com. 174 (1991), 96-101), does not result inany change in the ALP activity after incubation with conditioned mediumfrom L cell transfectants producing MP52 (HindIII-MP52/pABWN) or mediumafter MP52 production by infection with recombinant Vaccinia viruses.This indicates that MP52 has a cell specificity that partially deviatesfrom that of BMP2. Different functions due to different target sites forthe individual TGF-β family members may be of great medical relevance.

2. In Vivo Experiments

2.1

The most definitive possibility of examining bone development is basedon ectopic bone formation in vivo. This can for example be induced byimplantation of demineralized bone matrix (Urist, Science 150 (1965),893-899). The same process can be induced by combination of inactivematrix with bone-inducing proteins as described for example by Sampathet al. (PNAS* Proc.Natl.Acad.Sci. USA 78 (1981), 7599-7603). Thisprocess of bone formation is similar to embryonic enchondral boneformation and adult bone healing. This method therefore enables proteinsto be examined for their bone-inductive capability in vivo.

MP52 protein which had been obtained by expression in the vacciniasystem (see example 2) was partially purified and implanted for such anexperiment.

For this 143B cells (HuTk, ATCC CRL 8303) were cultured in culturedishes and roller flasks until confluence and infected with recombinantviruses as described in example 2 for the expression analyses, they werewashed and MP52 was allowed to accumulate for about 20 hours in MEM(Gibco BRL, ca 1 ml per 10⁶ cells). The same preparation was infectedwith wild-type viruses as a control. Cell culture supernatant(conditioned medium) from each preparation was collected and centrifuged(40000×g for 30 minutes at 4° C.). In order to remove the viruses, thesupernatants were filtered over inorganic filters (0.1 μm pore size,Whatman, Anotop 25). In the course of the characterization of MP52 itwas shown that this protein binds to heparin-Sepharose. This propertywas utilized for partial purification. For this the filtered andcentrifuged, conditioned medium was brought to a final concentration of50 mM Tris pH 7.0, 100 mM NaCl and 6 M urea and it was loaded onto aheparin column (HiTrap™, Pharmacia #17-0407-01) that uses equilibratedin buffer A (50 mM Tris pH 7.0, 100 mM NaCl and 6 M urea). The loadedcolumn was washed with buffer A and eluted with a linear gradient to100% buffer B (50 mM Tris pH 7.0, 600 mM NaCl and 6 M urea) at a flowrate of 0.5 ml/min within 50 min (2.5 ml per fraction). The use of ureais not absolutely necessary. MP52 elutes reproducibly mainly in 2fractions at about 250 to 400 mM NaCl as could be examined by Westernblot analysis (see example 2). Aliquots of

these fractions were also examined in 15% polyacrylamide gels stainedwith silver according to the instructions of the manufacturer (SilverStain-II, Daiichi #SE140000) and the fractions were pooled. Thecomparable fractions were also pooled after analysis in gels stainedwith silver after purification from conditioned medium after infectionwith wild-type viruses.

Further examinations on MP52 showed that MP52 also binds tohydroxyapatite. Thus it is in principle possible to achieve anadditional purification by a hydroxyapatite column or to replace aheparin column by a hydroxyapatite column (e.g. BIO-RAD, Econo-pac HTP).Other methods known to a person skilled in the art are also conceivablefor further purifications such as e.g. gel sieve columns, ion exchangercolumns, affinity columns, metal chelate columns or columns based onhydrophobic interactions.

The MP52 protein prepurified by heparin-Sepharose chromatography or thecorresponding proteins that are still contaminated which are alsopresent in the cell culture supernatants infected with the wild-type,were further purified by means of reversed phase HPLC. For this a C8column (Aquapore RP300, Applied Biosystems, particle size: 7 μm, poresize: 300 Å) was equilibrated with 10% buffer B (buffer A: 0.1%trifluoroacetic acid; buffer B: 90% acetonitrile, 0.1% trifluoroaceticacid). After loading the column with the pooled fractions containingMP52 from the heparin column it was extensively washed with 10% bufferB. The bound protein was eluted with the following gradient: 10 to 50%buffer B for 20 minutes and 50 to 100% buffer B for 50 minutes.Fractions of 500 μl were collected and analysed by Western blot as wellas with gels stained with silver. The MP52 protein elutes under theselected conditions in the range of about 55 to 65% acetonitrile. Thefractions containing MP52 were pooled. The same procedure was carriedout with the corresponding fractions from the control purification ofcell culture supernatant from cells infected with wild-type viruses.

Partially purified MP52 protein at a concentration estimated to be 50ng/ml according to Western blot analysis also showed a significantincrease in the ALP activity in ROB-C26 cells after three days ofincubation.

Partially purified MP52 protein or control protein from thecorresponding partially purified cell culture supernatants afterinfection with wild-type viruses were reconstituted with matrix andimplanted in rats in order to prove its capability for cartilage andbone formation.

In principle various matrix materials known to a person skilled in theart should be usable i.e. natural (also modified) and syntheticallyprepared matrices, however, biocompatible porous materials that can bebiologically degraded are preferred. In these experiments bone matrixfrom rats was used that had been prepared essentially in a similar wayto that described by. Sampath et al. (PNAS 80 (1983), 6591-6595). Therat bones (femur and tibia) were demineralized in 0.6 M HCl for 24 hoursand subsequently bone marrow that was still present was removed. Afterwashing with water and defatting for three hours in achloroform/methanol (1/1) mixture, the bones were air-dried, powderizedin a mill in a deep-frozen state and particle sizes between 400 and 1000μm were sieved out. Subsequently the matrix was extracted for 7 days atroom temperature in 4 M guanidinium HCl in the presence of proteaseinhibitors. After washing extensively with water, the matrix waslyophilized and stored at 4° C. Matrices treated in this way do not ontheir own show bone-inducing activity.

Protein can be combined with the extracted bone matrix by variousmethods known to a person skilled in the art. MP52 protein or controlprotein that had been purified by means of heparin-Sepharose as well asby reversed phase HPLC, was combined after elution in theacetonitrile/trifluoro-acetic acid solution with 25 mg matrix in eachcase per implant, mixed well, deep-frozen and lyophilized.

For the implantation of matrix-bound MP52, two ca. 3 months-old rats(Whistar) were used which had been anaesthetised by intramuscularinjection of an anaesthetic (0.2 ml Rompun (Bayer) mixed with 0.5 mlKetanest 50 (Parke Davis)) using 0.14 ml per 100 g body weight.Bilateral pockets were prepared in the abdominal muscles for theimplants (beneath the thorax, starting ca. 0.5 cm below the lowestcostal arch). The matrix-bound MP52 (ca. 2 to 4 μg as estimated byWestern blot) as well as the corresponding matrix-bound control proteinswere moistened using 0.9% saline solution (Delta Pharma) and introducedinto the muscular pockets. The muscular pockets as well as the necessaryskin incisions were subsequently sutured. The rats were immunosuppressedwith Cyclosporin A (Sandimmun).

After 18 or after 26 days the implants were removed from the rats andfixed for histological examinations. Since after 26 days the implantwith MP52 allowed the assumption that macroscopically bone formation hadalready occurred, this was embedded in methylmeth-acrylate in order toprepare thin sections, the other implants were embedded in paraffin.Mineralized cartilage and bone tissues are accentuated in black by meansof the von Kossa staining technique (Romeis, B.; “MikroskopischeTechnik”, Ed: Böck, P.; Urban and Schwarzenberg; Munich, Baltimore,Vienna (1989)). In the trichromium staining according to Masson-Goldner(Romeis, B.; “Mikroskopische Technik”, Ed: Böck, P.; Urban andSchwarzenberg; Munich, Baltimore, Vienna (1989)), mineralized bonetissue and collagen are stained bright green, osteoid is stained red andcytoplasm reddish-brown. Both staining techniques were used on implantsfrom both rats. In both experimental animals considerable formation ofcartilage and bone was detected in the implants containing MP52 usingboth staining techniques. The corresponding implants with controlprotein showed no formation whatsoever of cartilage or bone. The numberof cartilage precursors with chondrocytes and cartilage areas withinitial formation of extracellular matrix and its mineralization inconcentric circles is higher in the MP52 implant after 18 days than inthe one after 26 days. Mature bone tissue with vectorial osteoidformation as well as individual osteocytes in the bone is, however, alsodetectable in the implant after 18 days. In addition closed ossicles canbe observed with the onset of bone marrow formation. In the implantafter 26 days areas of cartilage with initial matrix formation andcalcification are also detectable, the portion of mineralized bonetissue stained green and having osteocytes and osteoid edges has,however, significantly increased. In this implant bone marrow formationtogether with the occurrence of isolated fat cells can also be detected.For illustration FIG. 5 shows the staining test of the bone material(according to von Kossa stain.) from the entire implant after 26 days. Asmall section of the same implant is shown in FIG. 6 after stainingaccording to Masson-Goldner. It shows active bone with an edging ofcuboidal osteoblasts and osteoid in which individual embeddedosteoblasts can be recognized. Furthermore individual osteocytes canalso be seen in the mineralized bone tissue (stained green in theoriginal preparation). The formation of bone marrow is also detectable.

The experiment shows that recombinantly produced MP52 alone, incombination with a matrix is capable of inducing enchondral boneformation.

2.2

In order to confirm the results, a further ectopic test for boneformation using MP52 L cell transformants was carried out. L cells(1×10⁶ cells) producing MP52 (transfected with HindIII-MP52/pABWN) andnon-producing (pABWN-transfected) L cells were injected into thebilateral thigh muscles of three male naked mice in each case. Allanimals were killed after three weeks, the thigh muscles were excisedand these were examined with low energy X-ray radiation as well ashistopathologically.

Analysis by X-ray radiation shows dense material at the injection sitesin the muscle tissue of all L cells producing MP52 as listed in Table 3.Simple cartilage formation and calcified cartilage formation could bedetermined in the muscles using histological examinations. These resultsalso confirm that MP52 can induce enchondral bone formation.

TABLE 3 Cells producing MP52 (HindIII- Control cells MP52/pABWN) (pABWN)dense material by 3/3 0/3 x-ray analysis chondrocytes by 3/3 0/3histology calcified cartilage 3/3 0/3 formation by histology

The experiments that were carried out confirm that MP52 proteinstimulates the formation of cartilage from undifferentiated mesenchymalcells as well as the differentiation and maturation of osteoblasts. Thisleads to enchondral bone formation which is similar to the inductioncascade in embryonic bone formation and bone healing of fractures.

The conditions stated in the experiments are to be looked upon as anillustration of the MP52 activity but not as limitation. The inventioncan also be examined and characterized in another form.

41 1 2703 DNA Homo sapiens misc_feature (1)..(2703) coding region andnon-translated regions for TGF-beta protein MP-52 1 ccatggcctcgaaagggcag cggtgatttt tttcacataa atatatcgca cttaaatgag 60 tttagacagcatgacatcag agagtaatta aattggtttg ggttggaatt ccgtttccaa 120 ttcctgagttcaggtttgta aaagattttt ctgagcacct gcaggcctgt gagtgtgtgt 180 gtgtgtgtgtgtgtgtgtgt gtgtgtgtga agtattttca ctggaaagga ttcaaaacta 240 gggggaaaaaaaaactggag cacacaggca gcattacgcc attcttcctt cttggaaaaa 300 tccctcagccttatacaagc ctccttcaag ccctcagtca gttgtgcagg agaaaggggg 360 cggttggctttctcctttca agaacgagtt attttcagct gctgactgga gacggtgcac 420 gtctggatacgagagcattt ccactatggg actggataca aacacacacc cggcagactt 480 caagagtctcagactgagga gaaagccttt ccttctgctg ctactgctgc tgccgctgct 540 tttgaaagtccactcctttc atggtttttc ctgccaaacc agaggcacct ttgctgctgc 600 cgctgttctctttggtgtca ttcagcggct ggccagagga tgagactccc caaactcctc 660 actttcttgctttggtacct ggcttggctg gacctggaat tcatctgcac tgtgttgggt 720 gcccctgacttgggccagag accccagggg accaggccag gattggccaa agcagaggcc 780 aaggagaggccccccctggc ccggaacgtc ttcaggccag ggggtcacag ctatggtggg 840 ggggccaccaatgccaatgc cagggcaaag ggaggcaccg ggcagacagg aggcctgaca 900 cagcccaagaaggatgaacc caaaaagctg ccccccagac cgggcggccc tgaacccaag 960 ccaggacaccctccccaaac aaggcaggct acagcccgga ctgtgacccc aaaaggacag 1020 cttcccggaggcaaggcacc cccaaaagca ggatctgtcc ccagctcctt cctgctgaag 1080 aaggccagggagcccgggcc cccacgagag cccaaggagc cgtttcgccc accccccatc 1140 acaccccacgagtacatgct ctcgctgtac aggacgctgt ccgatgctga cagaaaggga 1200 ggcaacagcagcgtgaagtt ggaggctggc ctggccaaca ccatcaccag ctttattgac 1260 aaagggcaagatgaccgagg tcccgtggtc aggaagcaga ggtacgtgtt tgacattagt 1320 gccctggagaaggatgggct gctgggggcc gagctgcgga tcttgcggaa gaagccctcg 1380 gacacggccaagccagcggc ccccggaggc gggcgggctg cccagctgaa gctgtccagc 1440 tgccccagcggccggcagcc ggcctccttg ctggatgtgc gctccgtgcc aggcctggac 1500 ggatctggctgggaggtgtt cgacatctgg aagctcttcc gaaactttaa gaactcggcc 1560 cagctgtgcctggagctgga ggcctgggaa cggggcaggg ccgtggacct ccgtggcctg 1620 ggcttcgaccgcgccgcccg gcaggtccac gagaaggccc tgttcctggt gtttggccgc 1680 accaagaaacgggacctgtt ctttaatgag attaaggccc gctctggcca ggacgataag 1740 accgtgtatgagtacctgtt cagccagcgg cgaaaacggc gggccccact ggccactcgc 1800 cagggcaagcgacccagcaa gaaccttaag gctcgctgca gtcggaaggc actgcatgtc 1860 aacttcaaggacatgggctg ggacgactgg atcatcgcac cccttgagta cgaggctttc 1920 cactgcgaggggctgtgcga gttcccattg cgctcccacc tggagcccac gaatcatgca 1980 gtcatccagaccctgatgaa ctccatggac cccgagtcca caccacccac ctgctgtgtg 2040 cccacgcggctgagtcccat cagcatcctc ttcattgact ctgccaacaa cgtggtgtat 2100 aagcagtatgaggacatggt cgtggagtcg tgtggctgca ggtagcagca ctggccctct 2160 gtcttcctgggtggcacatc ccaagagccc cttcctgcac tcctggaatc acagaggggt 2220 caggaagctgtggcaggagc atctacacag cttgggtgaa aggggattcc aataagcttg 2280 ctcgctctctgagtgtgact tgggctaaag gccccctttt atccacaagt tcccctggct 2340 gaggattgctgcccgtctgc tgatgtgacc agtggcaggc acaggtccag ggagacagac 2400 tctgaatgggactgagtccc aggaaacagt gctttccgat gagactcagc ccaccatttc 2460 tcctcacctgggccttctca gcctctggac tctcctaagc acctctcagg agagccacag 2520 gtgccactgcctcctcaaat cacatttgtg cctggtgact tcctgtccct gggacagttg 2580 agaagctgactgggcaagag tgggagagaa gaggagaggg cttggataga gttgaggagt 2640 gtgaggctgttagactgtta gatttaaatg tatattgatg agataaaaag caaaactgtg 2700 cct 2703 2501 PRT Homo sapiens DOMAIN (1)..(501) TGF-beta protein MP-52 precursor2 Met Arg Leu Pro Lys Leu Leu Thr Phe Leu Leu Trp Tyr Leu Ala Trp 1 5 1015 Leu Asp Leu Glu Phe Ile Cys Thr Val Leu Gly Ala Pro Asp Leu Gly 20 2530 Gln Arg Pro Gln Gly Thr Arg Pro Gly Leu Ala Lys Ala Glu Ala Lys 35 4045 Glu Arg Pro Pro Leu Ala Arg Asn Val Phe Arg Pro Gly Gly His Ser 50 5560 Tyr Gly Gly Gly Ala Thr Asn Ala Asn Ala Arg Ala Lys Gly Gly Thr 65 7075 80 Gly Gln Thr Gly Gly Leu Thr Gln Pro Lys Lys Asp Glu Pro Lys Lys 8590 95 Leu Pro Pro Arg Pro Gly Gly Pro Glu Pro Lys Pro Gly His Pro Pro100 105 110 Gln Thr Arg Gln Ala Thr Ala Arg Thr Val Thr Pro Lys Gly GlnLeu 115 120 125 Pro Gly Gly Lys Ala Pro Pro Lys Ala Gly Ser Val Pro SerSer Phe 130 135 140 Leu Leu Lys Lys Ala Arg Glu Pro Gly Pro Pro Arg GluPro Lys Glu 145 150 155 160 Pro Phe Arg Pro Pro Pro Ile Thr Pro His GluTyr Met Leu Ser Leu 165 170 175 Tyr Arg Thr Leu Ser Asp Ala Asp Arg LysGly Gly Asn Ser Ser Val 180 185 190 Lys Leu Glu Ala Gly Leu Ala Asn ThrIle Thr Ser Phe Ile Asp Lys 195 200 205 Gly Gln Asp Asp Arg Gly Pro ValVal Arg Lys Gln Arg Tyr Val Phe 210 215 220 Asp Ile Ser Ala Leu Glu LysAsp Gly Leu Leu Gly Ala Glu Leu Arg 225 230 235 240 Ile Leu Arg Lys LysPro Ser Asp Thr Ala Lys Pro Ala Ala Pro Gly 245 250 255 Gly Gly Arg AlaAla Gln Leu Lys Leu Ser Ser Cys Pro Ser Gly Arg 260 265 270 Gln Pro AlaSer Leu Leu Asp Val Arg Ser Val Pro Gly Leu Asp Gly 275 280 285 Ser GlyTrp Glu Val Phe Asp Ile Trp Lys Leu Phe Arg Asn Phe Lys 290 295 300 AsnSer Ala Gln Leu Cys Leu Glu Leu Glu Ala Trp Glu Arg Gly Arg 305 310 315320 Ala Val Asp Leu Arg Gly Leu Gly Phe Asp Arg Ala Ala Arg Gln Val 325330 335 His Glu Lys Ala Leu Phe Leu Val Phe Gly Arg Thr Lys Lys Arg Asp340 345 350 Leu Phe Phe Asn Glu Ile Lys Ala Arg Ser Gly Gln Asp Asp LysThr 355 360 365 Val Tyr Glu Tyr Leu Phe Ser Gln Arg Arg Lys Arg Arg AlaPro Leu 370 375 380 Ala Thr Arg Gln Gly Lys Arg Pro Ser Lys Asn Leu LysAla Arg Cys 385 390 395 400 Ser Arg Lys Ala Leu His Val Asn Phe Lys AspMet Gly Trp Asp Asp 405 410 415 Trp Ile Ile Ala Pro Leu Glu Tyr Glu AlaPhe His Cys Glu Gly Leu 420 425 430 Cys Glu Phe Pro Leu Arg Ser His LeuGlu Pro Thr Asn His Ala Val 435 440 445 Ile Gln Thr Leu Met Asn Ser MetAsp Pro Glu Ser Thr Pro Pro Thr 450 455 460 Cys Cys Val Pro Thr Arg LeuSer Pro Ile Ser Ile Leu Phe Ile Asp 465 470 475 480 Ser Ala Asn Asn ValVal Tyr Lys Gln Tyr Glu Asp Met Val Val Glu 485 490 495 Ser Cys Gly CysArg 500 3 24 DNA Artificial Sequence misc_feature (1)..(24) adapterprimer 3 agaattcgca tgccatggtc gacg 24 4 23 DNA Homo sapiensmisc_feature (1)..(23) MP-52 internal primer 4 cttgagtacg aggctttcca ctg23 5 24 DNA Artificial Sequence misc_feature (1)..(24) adapter primer 5attcgcatgc catggtcgac gaag 24 6 23 DNA Homo sapiens misc_feature(1)..(23) MP-52 internal primer 6 ggagcccacg aatcatgcag tca 23 7 23 DNAHomo sapiens misc_feature (1)..(23) MP-52 internal primer 7 acagcaggtgggtggtgtgg act 23 8 44 DNA Artificial Sequence misc_feature (1)..(44)primer composed of oligo dT and an adapter sequence 8 agaattcgcatgccatggtc gacgaagctt tttttttttt tttt 44 9 20 DNA Homo sapiensmisc_feature (1)..(20) MP-52 internal primer 9 ccagcagccc atccttctcc 2010 24 DNA Homo sapiens misc_feature (1)..(24) MP-52 internal primer 10tccagggcac taatgtcaaa cacg 24 11 24 DNA Homo sapiens misc_feature(1)..(24) MP-52 internal primer 11 actaatgtca aacacgtacc tctg 24 12 10DNA Artificial Sequence misc_feature (1)..(10) adapter 12 agcggccgct 1013 102 PRT Homo sapiens DOMAIN (1)..(102) partial sequence of MP-52starting with the first of the seven conserved cysteins 13 Cys Ser ArgLys Ala Leu His Val Asn Phe Lys Asp Met Gly Trp Asp 1 5 10 15 Asp TrpIle Ile Ala Pro Leu Glu Tyr Glu Ala Phe His Cys Glu Gly 20 25 30 Leu CysGlu Phe Pro Leu Arg Ser His Leu Glu Pro Thr Asn His Ala 35 40 45 Val IleGln Thr Leu Met Asn Ser Met Asp Pro Glu Ser Thr Pro Pro 50 55 60 Thr CysCys Val Pro Thr Arg Leu Ser Pro Ile Ser Ile Leu Phe Ile 65 70 75 80 AspSer Ala Asn Asn Val Val Tyr Lys Gln Tyr Glu Asp Met Val Val 85 90 95 GluSer Cys Gly Cys Arg 100 14 101 PRT Homo sapiens DOMAIN (1)..(101)portion of BMP 2 corresponding to MP 52 14 Cys Lys Arg His Pro Leu TyrVal Asp Phe Ser Asp Val Gly Trp Asn 1 5 10 15 Asp Trp Ile Val Ala ProPro Gly Tyr His Ala Phe Tyr Cys His Gly 20 25 30 Glu Cys Pro Phe Pro LeuAla Asp His Leu Asn Ser Thr Asn His Ala 35 40 45 Ile Val Gln Thr Leu ValAsn Ser Val Asn Ser Lys Ile Pro Lys Ala 50 55 60 Cys Cys Val Pro Thr GluLeu Ser Ala Ile Ser Met Leu Tyr Leu Asp 65 70 75 80 Glu Asn Glu Lys ValVal Leu Lys Asn Tyr Gln Asp Met Val Val Glu 85 90 95 Gly Cys Gly Cys Arg100 15 101 PRT Homo sapiens DOMAIN (1)..(101) portion of BMP 4corresponding to MP 52 15 Cys Arg Arg His Ser Leu Tyr Val Asp Phe SerAsp Val Gly Trp Asn 1 5 10 15 Asp Trp Ile Val Ala Pro Pro Gly Tyr GlnAla Phe Tyr Cys His Gly 20 25 30 Asp Cys Pro Phe Pro Leu Ala Asp His LeuAsn Ser Thr Asn His Ala 35 40 45 Ile Val Gln Thr Leu Val Asn Ser Val AsnSer Ser Ile Pro Lys Ala 50 55 60 Cys Cys Val Pro Thr Glu Leu Ser Ala IleSer Met Leu Tyr Leu Asp 65 70 75 80 Glu Tyr Asp Lys Val Val Leu Lys AsnTyr Gln Glu Met Val Val Glu 85 90 95 Gly Cys Gly Cys Arg 100 16 102 PRTHomo sapiens DOMAIN (1)..(102) portion of BMP 5 corresponding to MP 5216 Cys Lys Lys His Glu Leu Tyr Val Ser Phe Arg Asp Leu Gly Trp Gln 1 510 15 Asp Trp Ile Ile Ala Pro Glu Gly Tyr Ala Ala Phe Tyr Cys Asp Gly 2025 30 Glu Cys Ser Phe Pro Leu Asn Ala His Met Asn Ala Thr Asn His Ala 3540 45 Ile Val Gln Thr Leu Val His Leu Met Phe Pro Asp His Val Pro Lys 5055 60 Pro Cys Cys Ala Pro Thr Lys Leu Asn Ala Ile Ser Val Leu Tyr Phe 6570 75 80 Asp Asp Ser Ser Asn Val Ile Leu Lys Lys Tyr Arg Asn Met Val Val85 90 95 Arg Ser Cys Gly Cys His 100 17 102 PRT Homo sapiens DOMAIN(1)..(102) portion of BMP 6 corresponding to MP 52 17 Cys Arg Lys HisGlu Leu Tyr Val Ser Phe Gln Asp Leu Gly Trp Gln 1 5 10 15 Asp Trp IleIle Ala Pro Lys Gly Tyr Ala Ala Asn Tyr Cys Asp Gly 20 25 30 Glu Cys SerPhe Pro Leu Asn Ala His Met Asn Ala Thr Asn His Ala 35 40 45 Ile Val GlnThr Leu Val His Leu Met Asn Pro Glu Tyr Val Pro Lys 50 55 60 Pro Cys CysAla Pro Thr Lys Leu Asn Ala Ile Ser Val Leu Tyr Phe 65 70 75 80 Asp AspAsn Ser Asn Val Ile Leu Lys Lys Tyr Arg Asn Met Val Val 85 90 95 Arg AlaCys Gly Cys His 100 18 102 PRT Homo sapiens DOMAIN (1)..(102) portion ofBMP 7 corresponding to MP 52 18 Cys Lys Lys His Glu Leu Tyr Val Ser PheArg Asp Leu Gly Trp Gln 1 5 10 15 Asp Trp Ile Ile Ala Pro Glu Gly TyrAla Ala Tyr Tyr Cys Glu Gly 20 25 30 Glu Cys Ala Phe Pro Leu Asn Ser TyrMet Asn Ala Thr Asn His Ala 35 40 45 Ile Val Gln Thr Leu Val His Phe IleAsn Pro Glu Thr Val Pro Lys 50 55 60 Pro Cys Cys Ala Pro Thr Gln Leu AsnAla Ile Ser Val Leu Tyr Phe 65 70 75 80 Asp Asp Ser Ser Asn Val Ile LeuLys Lys Tyr Arg Asn Met Val Val 85 90 95 Arg Ala Cys Gly Cys His 100 1936 DNA Artificial Sequence misc_feature (1)..(36) primer OD 19atgaattccc atggacctgg gctggmakga mtggat 36 20 22 DNA Homo sapiensmisc_feature (1)..(22) portion of BMP 2 corresponding to primer OD 20acgtggggtg gaatgactgg at 22 21 22 DNA Homo sapiens misc_feature(1)..(22) portion of BMP 3 corresponding to primer OD 21 atattggctggagtgaatgg at 22 22 22 DNA Homo sapiens misc_feature (1)..(22) portionof BMP 4 corresponding to primer OD 22 atgtgggctg gaatgactgg at 22 23 22DNA Homo sapiens misc_feature (1)..(22) portion of BMP 7 correspondingto primer OD 23 acctgggctg gcaggactgg at 22 24 22 DNA Homo sapiensmisc_feature (1)..(22) portion of TGF-beta-1 corresponding to primer OD24 aggacctcgg ctggaagtgg at 22 25 22 DNA Homo sapiens misc_feature(1)..(22) portion of TGF-beta-2 corresponding to primer OD 25 gggatctagggtggaaatgg at 22 26 22 DNA Homo sapiens misc_feature (1)..(22) portionof TGF-beta-3 corresponding to primer OD 26 aggatctggg ctggaagtgg gt 2227 22 DNA Homo sapiens misc_feature (1)..(22) portion of Inhibin alphacorresponding to primer OD 27 agctgggctg ggaacggtgg at 22 28 22 DNA Homosapiens misc_feature (1)..(22) portion of Inhibin beta-A correspondingto primer OD 28 acatcggctg gaatgactgg at 22 29 22 DNA Homo sapiensmisc_feature (1)..(22) portion of Inhibin beta-B corresponding to primerOD 29 tcatcggctg gaacgactgg at 22 30 29 DNA Artificial Sequencemisc_feature (1)..(29) Primer OID 30 atgaattcga gctgcgtsgg srcacagca 2931 21 DNA Homo sapiens misc_feature (1)..(21) portion of BMP 2corresponding to primer OID 31 gagttctgtc gggacacagc a 21 32 21 DNA Homosapiens misc_feature (1)..(21) portion of BMP 3 corresponding to primerOID 32 catcttttct ggtacacagc a 21 33 21 DNA Homo sapiens misc_feature(1)..(21) portion of BMP 4 corresponding to primer OID 33 cagttcagtgggcacacaac a 21 34 21 DNA Homo sapiens misc_feature (1)..(21) portion ofBMP 7 corresponding to primer OID 34 gagctgcgtg ggcgcacagc a 21 35 21DNA Homo sapiens misc_feature (1)..(21) portion of TGF-beta-1corresponding to primer OID 35 cagcgcctgc ggcacgcagc a 21 36 21 DNA Homosapiens misc_feature (1)..(21) portion of TGF-beta-2 corresponding toprimer OID 36 taaatcttgg gacacgcagc a 21 37 21 DNA Homo sapiensmisc_feature (1)..(21) portion of TGF-beta-3 corresponding to primer OID37 caggtcctgg ggcacgcagc a 21 38 21 DNA Homo sapiens misc_feature(1)..(21) portion of Inhibin alpha corresponding to primer OID 38ccctgggaga gcagcacagc a 21 39 21 DNA Homo sapiens misc_feature (1)..(21)portion of Inhibin beta-A corresponding to primer OID 39 cagcttggtgggcacacagc a 21 40 21 DNA Homo sapiens misc_feature (1)..(21) portion ofInhibin beta-B corresponding to primer OID 40 cagcttggtg ggaatgcagc a 2141 10 DNA Artificial Sequence misc_feature (1)..(10) Adapter 41agcggccgct 10

What is claimed is:
 1. An isolated protein of the TGF-β family encodedby an isolated DNA molecule which comprises a sequence selected from thegroup consisting of: (a) the sequence shown in SEQ ID NO:1, (b) a partof SEQ ID NO:1 which encodes nucleotide 1783-2142 and encodes the matureprotein, (c) a nucleotide sequence which encodes the amino acid sequenceaccording to SEQ ID NO: 2, (d) a nucleotide sequence which encodes themature protein with amino acids 382-501 according to SEQ ID NO:2.
 2. Theisolated protein as claimed in claim 1, wherein the protein has theamino acid sequence shown in SEQ ID NO. 2, the mature part thereof orthe mature part combined with the propeptide part of SEQ ID NO:2.
 3. Apharmaceutical composition comprising at least one protein according toclaim
 1. 4. The pharmaceutical composition as claimed in claim 3,wherein the pharmaceutical composition is suitable for the treatment ofbone, cartilage or connective tissues or teeth.
 5. The pharmaceuticalcomposition as claimed in claim 3, wherein the pharmaceuticalcomposition is suitable for applications in connection withangiogenesis.
 6. A pharmaceutical composition according to claim 3,further comprising a pharmaceutically acceptable carrier, diluent orfiller.
 7. A dental implant which contains the protein according toclaim
 1. 8. An isolated protein of the TGF-β family encoded by a DNAmolecule which comprises the part of SEQ ID NO:1 which encodes themature protein according to claim 1 and further comprises the part ofSEQ ID NO:1 coding for at least one of signal peptide or propeptide. 9.A pharmaceutical composition comprising at least one protein accordingto claim 1, wherein the protein is contained on and/or in a matrixmaterial.
 10. A pharmaceutical composition according to claim 9, whereinsaid matrix material is a biocompatible porous material that can bebiologically degraded.
 11. A protein according to claim 1, wherein theprotein is a dimer.
 12. An isolated protein of the TGF-β family encodedby a DNA molecule which comprises nucleotides 640-2142 of SEQ ID NO:1.